Leishmaniasis is a major and severe parasitic disease that affects humans, canines, and to a lesser degree, felines.
Leishmania and Viannia subgenera are grouped into complexes of species and subspecies based upon molecular, biochemical and immunological similarities. There are several forms of the disease named by their clinical presentation including cutaneous, mucocutaneous or visceral leishmaniasis. Each of these forms of disease is caused by different species of sand flies found in different regions of the world. Cutaneous leishmaniasis of humans is associated with members of L. aethiopica, L. major, and L. tropica complexes in the Old World and L. mexicana and L. braziliensis complexes in the New World. Visceral leishmaniasis is caused by L. donovani and L. infantum in Old World regions while L. chagasi is primarily responsible for visceral disease in the New World. Because L. infantum is the primary agent associated with canine leishmaniasis, infections in dogs often are regarded as visceral even though they tend to cause both visceral and cutaneous disease.
The agent of visceral leishmaniasis is a protozoan parasite and belongs to the leishmania donovani complex. This parasite is widely distributed in temperate and subtropical countries of Southern Europe, Africa, Asia, South America and Central America (Desjeux P. et al., 1984, Nucl. Acids Res., 12:387-395). Leishmania donovani infantum (L. infantum) is responsible for the feline and canine disease in Southern Europe, Africa, and Asia. In South America and Central America, the agent is Leishmania donovani chagasi (L. chagasi), which is closely related to L. infantum. In humans, the agent is Leishmania donovani donovani (L. donovani), which is also related to L. infantum and L. chagasi. 
Leishmaniasis is a slowly progressive disease that can take up to 7 years to become clinically apparent (McConkey S E et al., 2002, Canine Vet J 43:607-609). Even then, signs are frequently nonspecific and a diagnosis of Leishmania is seldomly considered. Dogs are most commonly infected with L. infantum (L. donovani complex) which is responsible for viscerotropic disease in people. However, up to 90% of infected dogs present with both visceral and cutaneous lesions (Slappendel R J et al., 1998, In: Greene C E: Infectious Diseases of the Dog and Cat, pp 450-458). On the other hand, many dogs appear naturally resistant to this parasite and may remain asymptomatic despite known infection (Grosjean N L et al., 2002, Vet Rec 150:241-244). It is estimated that only 10% of dogs residing in endemic areas actually develop clinical disease (Lindsay D S et al., 2002, Compend Cont Educ Pract Vet 24:304-312). This lower incidence of clinical disease is attributed to a genetic predisposition of certain dogs to mount a more protective cell-mediated immune response than a humoral response (Lindsay D S et al., McConkey S E et al., Slappendel R J, et al.). Furthermore, it has been reported that up to 20% of infected dogs may mount an adequate immune response and spontaneously recover from clinical illness (McConkey S E et al.). In animals that mount a humoral response, IgG1 appears to correlate with clinical disease while asymptomatic dogs have higher IgG2 antibody levels (Lindsay et al.).
Some of the more frequently reported clinical signs of leishmaniasis include listlessness, fatigue and exercise intolerance coupled with anorexia and weight loss that eventually culminate as wasting disease (McConkey S E et al.). These signs may or may not be accompanied by fever, local or generalized lymphadenopathy (90%) and/or hepatosplenomegaly (Grosjean N L et al., 2003, J Am Vet Med Assoc 222:603-606; Lindsay D S et al., McConkey S E et al.; Martinez-Subiela S et al., 2002, Vet Rec 150:241-244). Articular involvement is also fairly common and may present as lameness with swollen joints or simply as a stiff gait. Less common findings include ocular lesions (<5%), chronic diarrhea (30%) and long, deformed brittle nails (20%) referred to as onychogryphosis (Lindsay D S et al., Slappendel R J et al.). Cutaneous lesions are present in up to 89% of infected dogs, with or without overt signs of visceral involvement. Lesions of cutaneous leishmaniasis may occur anywhere on the body but the most common sites are those which are exposed to the environment and are therefore more susceptible to bites from the sand flies. The initial papule rapidly gives rise to an ulcer. Viseral leishmaniasis is invariably fatal if not treated promptly. Viseral leishmaniasis affects the internal body organs, specifically the spleen and the liver.
Dogs are considered the major reservoir of Leishmaniasis. The disease is characterized by chronic evolution of viscero-cutaneous signs occurring in less than 50% of infected animals (Lanotte G. et al., 1979, Ann. Parasitol. Hum. Comp. 54:277-95). Both asymptomatic and symptomatic dogs with detectable antibodies may be infectious (Molina R. et al., 1994, Trans. R. Soc. Med. Hyg. 88:491-3; Courtenay O. et al., 2002, J. Infect. Dis., 186:1314-20). Cats may also be carriers of the protozoan parasites and are thus considered secondary potential reservoirs.
Due to a number of factors, treatment options for leishmaniasis in dogs and response to therapy are limited at best. For some undefined reason, visceral leishmaniasis is more difficult to treat in dogs than in humans. No treatment option is 100% effective in clearing parasitic infection and clinical disease often reappears with cessation of therapy (Lindsay D S et al.). In endemic areas, the most common treatment regimen has been a combination of allopurinol with a pentavalent antimonial such as meglumine antimonite or sodium stibogluconate (Lindsay D S et al., Slappendel R J et al.). However, in recent years this protocol has fallen out of favor due to increasing resistance of the parasite to the drug as well as adverse side effects associated with these compounds (Lindsay D S et al.). To further limit treatment options, PENTOSTAM® (sodium stibogluconate) is the only available antimonial in the United States and its distribution is regulated by the Centers for Disease Control and Prevention (CDC) in Atlanta, Ga. (Lindsay D S et al.). Other investigations have sought to identity methods of preventing and treating leishmaniasis through, for example, administration of antigenic fusion polypeptides (see US 2009/0291099 which is hereby incorporated herein by reference in its entirety).
Different protocols have been tried but have proven no more efficacious at clearing parasitic infection or at preventing clinical relapse. In addition, each protocol is associated with potential adverse effects. Amphotericin B binds sterols and disrupts cell membrane permeability but is nephrotoxic (Lindsay D S et al.). When given parenterally, Paramomycin acts synergistically with antimonials causing higher levels of the antimonial for longer periods of time but is also nephrotoxic and is not currently recommended for clinical use (Lindsay D S et al.). Pentamidine isethionate is effective against leishmaniasis but requires at least 15 intramuscular injections and is quite painful (Lindsay D S et al.). Ketaconazole, miconazole, fluconazole and itraconazole are oral drugs that may be useful in containing the disease but are cost prohibitive and carry the risk of drug resistance when treating patients symptomatically. In summary, the various treatment regimens for leishmaniasis in dogs have been investigated but are not 100% efficacious; relapses are the rule rather than the exception. Ultimately, the veterinary practitioner is faced with the dilemma of treating symptomatic outbreaks of leishmaniasis in dogs at the risk of developing drug resistant strains of this parasite within the United States.
Mass detection of seropositive dogs followed by culling and/or drug treatment, or the mass application of deltamethrin-impregnated collars, was shown to have an impact in reducing human and canine Leishmaniasis prevalence in endemic areas of Southern Europe, Africa, and Asia (Maroli M. et al., 2001, Med. Vet. Entomol. 15:358-63; Mazloumi Gavgani A. S. et al., 2002, Lancet 360:374-9), although the efficacy of eliminating seropositive canines has been debated (Dietze R. et al., 1997, Clin. Infect. Dis. 25:1240-2; Moreira Jr. E. D. et al., 2004, Vet. Parasitol. 122:245-52). These control measures are either considered unacceptable, expensive or not effective (Gradoni L. et al., 2005, Vaccine 23:5245-51).
Mathematical models used to compare the effectiveness of various tools for controlling Leishmaniasis suggest that a canine vaccine may be the most practical and effective method (Dye C., 1996, Am. J. Trop. Med. Hyg. 55:125-30). Therefore, the development of vaccines able to protect canines from Leishmaniasis and/or to prevent disease progression in infected animals is highly desirable for the implementation of Leishmaniasis control programs as well for the veterinary community (Gradoni L. et al.).
Haynes et al. (Biotechnol. Prog., 2010, Vol. 26, No. 3, 743-749) discuss the use of high hydrostatic pressure to achieve high solubility and high refolding yields of growth hormone (GH) produced in E. coli inclusion bodies. U.S. Pat. Nos. 6,489,450, 7,064,192, 7,767,795 and 7,615,617 disclose reversing aggregation and increasing refolding of denatured proteins by application of high pressure.
There remains a need for effective and efficient methods of producing subunit (protein) vaccine for the treatment of Leishmania. The vaccine formulation and the method of producing such vaccine of the present invention fulfill this long felt need in the art.